In recent years, formulations containing proteins and peptides as active ingredients have been developed as a result of advancement of genetic recombination and chemical synthesis technologies, and the number of such formulations is increasing every year. However, proteins and peptides are not easily absorbed from the gastrointestinal tract, mucous membrane, etc. Furthermore, proteins and peptides are unstable in the body, and have a short half-life in blood. Hence, protein formulations and peptide formulations must be administered by injection repeatedly at frequent intervals, thus imposing excessive burdens on patients as well as medical staff. There is therefore a demand for a sustained-release DDS matrix for encapsulating a protein or peptide without impairing pharmacological activity. Further, in terms of administration efficiency, it is desirable to encapsulate as much protein and/or peptide as possible into a matrix.
It is known that the pharmacological activity of proteins and peptides largely depends on their higher-order structures, and is impaired by denaturation and aggregation arising from external environments such as contact with an organic solvent or air interface, pressure, temperature, and pH. It is also known that administration of a denatured or aggregated protein into the body increases risks of antigenicity and the like. As to a sustained-release formulation having a protein or peptide as an active ingredient, it is required to ensure stability of the protein or peptide during a step of producing the formulation, storage of the formulation, and until the release of the active ingredient in the body after administration.
Attempts have been made to prepare practical sustained-release formulations based on a biodegradable polymer matrix such as a polylactic acid-polyglycolic acid copolymer (PLGA), but such formulations are reported to cause protein denaturation or aggregation due to matrix hydrophobicity and as a result of manipulations required for formulation (e.g., emulsification, drying, acidification) (Non-patent Documents 1 and 2).
On the other hand, there are also reports of sustained-release formulations based on a hydrophilic hydrogel matrix, but no such formulations are yet available for practical application due to problems of protein stability during a gelling step, etc.
In terms of safety, matrices used for formulations should have non-antigenicity, non-mutagenicity, non-toxicity, and biodegradability. There is not known any sustained-release formulation ready for practical use in all aspects, i.e., encapsulation amount and recovery ratio of proteins or peptides, as well as safety.
Some recent reports have proposed the use of polysaccharides as matrixes for drug carriers. Among them, hyaluronic acid (HA), a biomaterial (polysaccharide) isolated from the vitreous body of bovine eyes in 1934 by K. Meyer, has been known as a major component of extracellular matrix for a long time. HA is a kind of glycosaminoglycan composed of disaccharide unit(s) in which D-glucuronic acid and N-acetylglucosamine are linked to one another via β(1→3) glycosidic linkages. There is no difference among species in the chemical and physical structure of HA and humans also have a metabolic system for HA. HA is therefore one of the safest medical biomaterials in terms of immunity and toxicity. Recent years have enabled microbial mass production of high-molecular-weight HA and also have allowed practical use of HA in the fields of therapeutic agents for degenerated cartilage, cosmetics, etc.
Having non-antigenicity, non-mutagenicity, non-toxicity, and biodegradability, HA appears to be preferred as a matrix of a sustained-release formulation in terms of safety. To date, there have been many reports of formulations using HA as a matrix; use of modified HA for the purpose to prolong residence time in blood (Patent Document 1), use of an alkyl chain-introduced HA derivative for the purpose to prolong residence time in the knee joint (Patent Document 2), use of in situ crosslinked HA gel for sustained release of a protein (Patent Document 3), and use of a hyaluronic acid ester solid for sustained release of a bone morphogenetic protein (BMP) (Patent Document 4) have been reported.
On the other hand, there are several reports of matrices that are spontaneously complexed with a protein or peptide in an aqueous solution in the absence of an organic solvent, and such matrices are produced using, mainly, polysaccharide or polyamino acid as a raw material.
As an exemplary case of a pharmaceutical formulation using a polyamino acid derivative as a matrix, the use of a tocopherol-introduced polyglutamic acid matrix is reported (Patent Document 5).
With regard to a polysaccharide derivative matrix, it is reported that a pullulan derivative in which a cholesteryl group or the like is introduced forms a nano-size particle in an aqueous solution, and serves as a host molecule which is complexed with a hydrophobic low-molecular-weight substance, a peptide, a protein or the like (Non-patent Documents 3 to 10). A thermodynamic evaluation of the pullulan derivative after protein incorporation shows that the incorporated protein forms a hydrogen bond with a hydroxy group of pullulan and is thereby stabilized (Non-patent Document 11).
There is also a report of the use of carboxymethyl cellulose (CMC) (Patent Document 6) and linoleic acid-introduced chitosan (Non-patent Document 12) as raw materials for forming a complex with a protein. Furthermore, Patent Document 8, which was published after the priority date of the subject application, reports a composition comprising a hyaluronic acid derivative having a crosslinkable group and a hydrophilic polysaccharide derivative having a hydrophobic group, wherein the hyaluronic acid derivative having a crosslinkable group is prepared by the bridge-formation reaction of hyaluronic acid or a derivative thereof having a crosslinkable group in the presence of the hydrophilic polysaccharide derivative.
It is reported that HA receptors such as CD44, RHAMM (receptor for hyaluronic acid-mediated motility), LYVE-1 (lymphe vessel endothelial HA receptor-1), and HARE (hyaluronic acid receptor for endocytosis) are present in the body (Non-patent Documents 18 and 19). Especially CD44 and RHAMM are overexpressed in many cancer cells. Thus, attempts have been made to use HA as a cancer targeting carrier matrix. Examples thereof include paclitaxel-HA conjugates (Non-patent Documents 20 to 22 and Patent Document 9), camptothecin-HA conjugates (Patent Document 10), doxorubicin-HPMA[N-(2-hydroxypropyl)methacrylamide]-HA conjugates (Non-patent Document 23), butyric acid-HA conjugates (Non-patent Document 24), doxorubicin-encapsulating HA-PEG-PLGA nanoparticles (Non-patent Document 25), siRNA-encapsulating HA gels (Non-patent Document 26), doxorubicin-encapsulating HA-coated liposomes (Non-patent Document 27), etc. Non-patent Document 28, which was published after the priority date of the subject application, reports HA derivatives conjugated to cholic acid via an ethylenediamine linker introduced via an amide bond. It is reported that carriers having the above HA matrices are efficiently incorporated into CD44-hyperexpressed cells in vitro (e.g., Non-patent Document 20). However, it is known that when systemically administered, HA is instantaneously incorporated into HARE receptors present in sinusoidal endothelium such as liver and then metabolized, and it is eliminated rapidly from blood (Non-patent Documents 29 to 31). Thus, to achieve efficient cancer targeting using HA matrices, a carrier with reduced uptake by liver and prolonged residence time in blood is needed.